Differential Diagnosis Of Cancer And Other Conditions Based On Expression Of p63

ABSTRACT

This invention relates to methods of distinguishing among various types of differentiated and undifferentiated epithelial carcinomas, and non-epithelial carcinomas, by detecting the presence of p63 nucleic acid or protein expression. The invention also provides methods for detecting p63 nucleic acids and proteins, as well as methods for diagnosing and treating certain tumors based on whether the tumors express p63.

GOVERNMENT SUPPORT This invention was made with government support undergrant R21-CA81362 awarded by the National Cancer Institute. Thegovernment has certain rights in the invention. FIELD OF THE INVENTION

The present invention relates to a gene product, referred to herein asp63, and to nucleic acids, including the gene, mRNAs, and cDNAs,encoding it, in particular, to methods of using the gene and geneproduct to diagnose and/or treat certain diseases and disorders such ascancer. The invention also relates to kits which may be used in suchdiagnostic and treatment methods

BACKGROUND OF THE INVENTION

The nuclear protein p63, located on chromosome 3q, has been shown topossess homology with p53, a tumor-suppressor gene encoding amulti-functional DNA-binding protein important in cell cycle and celldeath regulation. p63 exists in three isoforms, each of which can encodetwo categories of transcripts under the control of two alternativepromoters. The first encodes full length proteins with an N-terminaltransactivation domain that, like p53, can activate transcription andinduce apoptosis. The second encodes truncated proteins lacking theN-terminal transactivation domain (ΔNp63), and potentially acting in adominant-negative manner to suppress transactivation by p53 and/or fulllength p63.

Little is known about the function of p63. Evidence suggests that p63 isnecessary for normal development (Levrero et al., J. Cell Sci. 2000,113:1661-70). Immunohistochemical studies using an anti-p63 polyclonalantiserum show that the protein is widely detectable, with predominantassociation in the proliferative compartments in epithelia but also innon-proliferative populations (Hall et al., Carcinogenesis 2000,21:153-60). Normal human epidermis, hair follicles, and stratifiedepidermal cultures show p63 expression primarily in cells withproliferative potential, particularly basal cells, and not in cellsundergoing terminal differentiation (Parsa et al., J. Invest, Dermatol.1999, 113:1099-1105). The p63 RNA present in the basal cells was mainlytruncated, potentially dominant-negative isotypes (Parsa et al., supra).

Investigators have studied p63 expression in neoplasms. In keratinocytesquamous cell carcinomas, the number of cells containing p63 and theirdistribution depends on the degree of anaplasia (Parsa et al., supra).However, p63 does not yet have a defined role in neoplasia and otherphysiological and pathological situations (Hall et al., supra). Thus,there is a need in the art to determine what, if any role p63 plays inneoplasms and cancer.

SUMMARY OF THE INVENTION

The present invention are methods for detecting p63 nucleic acid andprotein expression in a cell or tissue that can be used for diagnosticand prognostic purposes. Such methods include nucleic acidhybridization. PCR and RT-PCR, and various protein assays employingantibodies that bind to p63 polypeptides. In a preferred embodiment, theprotein assay immunohistochemical staining using the anti-p63 4A4monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.). The present invention also provides a kit comprising thereagents required for detection of p63 nucleic acids or polypeptides.

The present invention also provides methods for distinguishingdifferentiated lung cancers from undifferentiated lung cancers byevaluating lung tissue for the expression of p63 nucleic acids orproteins. In particular, the expression of p63 indicates that the lungcancer is a differentiated carcinoma and absence of p63 expression isindicative of a undifferentiated carcinoma.

In a preferred embodiment, the undifferentiated lung cancer is a smallcell undifferentiated carcinoma and the differentiated lung cancer is apoorly differentiated squamous cell carcinoma, a moderatelydifferentiated squamous cell carcinoma, a well-differentiated squamouscell carcinoma, an adenosquamous carcinoma or an adenocarcinoma,

The present invention also provides methods of distinguishing anepithelial squamous cell carcinoma from a non-epithelial cell carcinomaby detecting p63 nucleic acid or protein expression in cells derivedfrom a carcinoma. In particular, p63 expression indicates that thecarcinoma is an epithelial squamous cell carcinoma and the absence ofp63 expression indicates that the carcinoma is a non-epithelial cellcarcinoma or a carcinoma without squamous differentiation potential.

In a preferred embodiment, the p63 positive cells are squamousepithelial cells, transitional cells or glandular epithelial cells andthe p63 negative cells are glandular carcinoma cells, or mostpreferably, renal carcinoma cells.

Also provided by the present invention is a method for distinguishing athyroid papillary carcinoma from another thyroid disorder in thyroidcells derived from a neoplasm, nodule, or enlargement by detecting p63nucleic acid or protein expression. p63 expression is indicative of apapillary carcinoma and absence of p63 expression is indicative of afollicular adenomata, a medullary carcinoma, an anaplastic carcinoma ora Hurthle cell carcinoma.

The present invention also provides a method of distinguishingHashimoto's thyroiditis from another inflammatory thyroid condition bydetecting p63 nucleic acid or protein expression in inflammatory thyroidcell. The presence p63 nucleic acid or protein expression indicates thatthe pathology is Hashimoto's thyroiditis and the absence of p63expression indicates that the inflammatory pathology is Grave's disease.

The present invention additionally provides a method for treating lungcancer in a patient by diagnosing the lung cancer as a squamous cellcarcinoma by detecting p63expression, followed by surgical resection ofthe p63-expressing tumor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for differentiallydistinguishing various types of cancer and other pathologicalconditions. For example, the invention permits distinguishing a specifictype of lung cancer, such as a poorly differentiated squamous cellcarcinoma, a moderately differentiated squamous carcinoma, a welldifferentiated squamous cell carcinoma, an adenosquamous carcinoma, andan adenocarcinoma, from an undifferentiated lung cancer, such as a smallcell undifferentiated carcinoma (oat cell carcinoma). Alternatively, theinvention provides for distinguishing papillary thyroid carcinoma fromother thyroid neoplasms. In yet another embodiment, the inventionprovides for distinguishing transitional cell carcinomas (from thekidney and ureter) from renal cell carcinomas. P63 detection may beemployed in any differential diagnosis between a tumor of squamousorigin, differentiation or squamous potential, and a tumor withoutsquamous differentiation or potential.

In another aspect, the invention permits detection or diagnosis ofprecancerous dysplasia, intra-epithelial neoplasms, or cancer(malignancy) in epithelial cells by detecting p63 expression in surface(not basal stem) epithelial cells, which ordinarily lack p63 expression.For example, cervical cells obtained by non-traumatic brushing or lavagethat express p63 are likely in a state of pre-malignancy or have becomemalignant. Other cell types amenable to this diagnostic approachinclude, but are not limited to, squamous epithelia (e.g., mouth,esophagus, trachea, anus); transitional cells (e.g., bladder, ureter,urethra, epithelia lining the respiratory tract); glandular epithelialcells (e.g., thyroid, endometrium, endocervix); and other cells that areor have the capacity to become squamous. This aspect of the inventionyields better results when the basal stem cells, that continuouslydivide to reform the epithelial tissue, are not assayed, since thesecells express p63 endogenously.

In each case, the method comprises detecting p63 expression in cellsfrom the cancer or other condition. The invention is based, in part, onthe observation that tumors or conditions arising from non-squamousepithelia having the capacity to undergo squamous differentiation arecapable of expressing p63. In contrast, tumors that never undergosquamous differentiation do not express p63.

In the case of cancer, p63 expression indicates that the lung cancer isa differentiated lung cancer. The absence of p63 expression indicatesthat the lung cancer is likely an undifferentiated lung cancer. Theinvention specifically provides for distinguishing poorly differentiatedsquamous carcinoma (p63 expression) from small cell undifferentiatedcarcinoma (no p63 expression), In another embodiment, the inventionprovides for distinguishing bronchioloalveolar carcinoma (no p63expression) from adenocarcinoma with alveolar spread pattern (p63expression). The invention further provides for diagnosing a new subtypeof adenosquamous carcinoma marked by basal p63 staining.

In the case of distinguishing between transitional cell and renal cellcancer, p63 expression indicates that the renal cancer is a transitionalcell carcinoma, and the absence of p63 expression indicates that therenal cancer is a renal cell carcinoma. For example, the inventionprovides for distinguishing upper uretal or renal pelvic transitionalcarcinoma (urothelial carcinoma), in which cells express p63, from renalcell carcinoma or adrenal carcinoma, in which the cells are negative forp63 expression.

In the case of thyroid cancer, p63 expression indicates that the thyroidcancer is a papillary thyroid carcinoma, and the absence of p63expression indicates that the thyroid cancer is a different thyroidneoplasm. In particular, the invention provides for distinguishingpapillary carcinoma, in which cells are positive for p63 expression,from follicular carcinoma, medullary carcinoma, follicular adenoma,Hurthle cell adenoma, Hurthle cell carcinoma, and nodular goiter inwhich cells are negative for p63 expression.

In addition, the invention advantageously provides for differentiationof non-cancerous thyroid conditions. In particular, it provides fordiagnosing antibody-positive and antibody-negative Hashimoto'sthyroiditis. This is particularly advantageous in the case ofantibody-negative Hashimoto's. It further permits distinguishingHashimoto's thyroiditis (p63 positive) from other inflammatory thyroidconditions, such as Grave's disease (which is p63 negative).

Various methods are available to detect p63 expression, includingbiochemical assays, immunoassays, Northern and reverse-transcriptasepolymerase chain reaction (RT-PCR) assays, and the like. In a specificembodiment, detecting p63 expression comprises detecting expression ofp63 protein. More particularly, detecting p63 protein expressioncomprises detecting the p63 protein with an immunoassay. In specificembodiments exemplified infra, the immunoassay is an immunohistochemicalassay or an immunocytopathology assay.

The assays of the invention can be performed on various cell samples.For example, tumor cell tissue obtained by biopsy, resection, or othertechnique can be tested. Alternatively, cytological samples can betested.

The invention further provides a method of treatment of lung cancer in apatient. The method comprises administering a chemotherapeutic agent toa patient diagnosed with a small cell undifferentiated carcinoma lungcancer, wherein the small cell undifferentiated carcinoma isdistinguished from a squamous cell carcinoma by detecting an absence ofp63 expression in cells from the lung cancer. In another embodiment, themethod comprises surgically resecting a squamous cell carcinoma from alung of a patient diagnosed with squamous cell carcinoma lung cancer,wherein the squamous cell carcinoma is distinguished from a small cellcarcinoma by detecting p63 expression in cells from the lung cancer.

As used herein, the term “p63” refers to a protein homologous to thetumor suppressor protein p53, which contains a multi-functionalDNA-binding protein important in cell cycle and cell death regulation.p63 exists in three isoforms, each of which can encode two categories oftranscripts under the control of two alternative promoters. The firstencodes full length proteins with an acidic N-terminal transactivationdomain that, like p53, can activate transcription and induce apoptosis.The second encodes truncated proteins lacking the N-terminaltransactivation domain (ΔNp63), and potentially acting in adominant-negative mariner to suppress transactivation by p53 and fulllength p63. Unless otherwise specified, p63 means any of the threeisoforms. The three p63 splice forms, α, β, and γ may be differentiallyexpressed in tumors.

A tumor is “p63 positive” or has detectable p63 expression when anycells in the tumor express p63. A tumor is “p63 negative” or has nodetectable p63 expression when no cells express p63 at detectablelevels.

As used herein, the term “cancer” refers to any malignant tumor,particularly arising in the lung, kidney, or thyroid. The cancermanifests itself as a “tumor” or tissue comprising malignant cells ofthe cancer. Examples of tumors that can be detected, diagnosed andtreated according to the invention include sarcomas and carcinomas suchas, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinorna, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hernangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. As noted above, the invention specifically pennitsdifferential diagnosis of lung, kidney, and thyroid tumors.

In particular, the invention permits differential diagnosis of variouslung cancers. These include poorly differentiated, moderatelydifferentiated, and high differentiated squamous cell carcinomas;bronchioloalveolar carcinoma, carcinoid, and small cell undifferentiatedcarcinoma. Bronchioloalveolar carcinoma is a well-differentiatedadenocarcinoma probably derived from lung alveolar epithelium. Carcinoidis a generally much less aggressive form of tumor than small cellundifferentiated, Small cell undifferentiated carcinoma is a highlyaggressive cancer.

The present invention also advantageously provides for assessing thetumorigenic potential of thyroid nodules. At present, detection ofthyroid nodules leads to fine needle aspiration to obtain cells from thenodule. Cytologic analysis of these cells can provide an indication ofwhether or not they are neoplastic or malignant. For example, follicularadenomas and papillary carcinoma may present as thyroid nodules. Fineneedle aspiration can fail to yield cells amenable to definitivepathological analysis. Detecting p63 expression in nodule cells deemedneoplastic permits a confident diagnosis of papillary carcinoma; theabsence of p63 expression leads to a diagnosis of a different pathology,such as follicular adenoma.

Accuracy in the pre-surgical determination of malignancy in thyroidnodules is essential, in order to avoid unnecessary surgery fornon-malignant thyroid conditions presenting as a nodule or enlargement.Although pre-surgical testing including ultrasonographic scans and fineneedle aspiration cytologic evaluation is useful for definitivelyidentifying some of these lesions pre-surgically, in many cases, thedefinitive decision to perform surgery or to choose the appropriatesurgery cannot be made.

For example, papillary thyroid carcinoma may have cytologic features ontine needle aspiration which are considered abnormal or suspicious, butnon-definitive, or may have features which are indistinguishable frombenign entities such as follicular adenomas. In these and othercircumstances, detection and evaluation of p63 at the gene, mRNA, orprotein level will allow for definitive diagnosis, reducing eitherunnecessary surgery for benign entities, inappropriate delays of monthsto years tor surgical removal of malignancy, and reduce the need forsecond surgical procedures, such as when a papillary carcinoma ismisdiagnosed as a benign adenoma, p63 may be detected alone, or in amultiplex panel with non-p63 probes, e.g., other antibodies, on a DNA orproteomics chip.

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can he free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. An isolatedorganelle, cell, or tissue is removed from the anatomical site in whichit is found in an organism. An isolated material may be, but need notbe, purified. The term “purified” as used herein refers to material thathas been isolated under conditions that reduce or eliminate the presenceof unrelated materials, i.e., contaminants, including native materialsfrom which the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography (includingpreparative solid phase chromatography, oligonucleotide hybridization,and triple helix chromatography), ultracentrifugation, and other means.Polypeptides and proteins can be purified by various methods including,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange andpartition chromatography, precipitation and salting-out chromatography,extraction, and countercurrent distribution. For some purposes, it ispreferable to produce the polypeptide in a recombinant system in whichthe protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence, ora sequence that specifically binds to an antibody, such as FLAG and GST.The polypeptide can then be purified from a crude lysate of the hostcell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the protein or againstpeptides derived therefrom can be used as purification reagents. Cellscan be purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting [FACS]). Other purification methods are possible. Apurified material may contain less than about 50%, preferably less thanabout 75%, and most preferably less than about 90%, of the cellularcomponents with which it was originally associated. The “substantiallypure” indicates the highest degree of purity which can be achieved usingconventional purification techniques known in the art.

A “sample” as used herein refers to a biological material which can betested, e.g., for the presence of p63 polypeptides or p63 nucleic acids,e.g., to identify cells that specifically express the p63 gene and itsgene product. Such samples can be obtained from any source, includingtissue biopsies, blood and blood cells, pleural effusions, pericardialeffussions, cerebrospinal fluid (CSF), urine, ascites fluid, cystfluids, bronchial aspiration, bronchoscopic washes and lavages,peritoneal washes, brushes and brush specimens, fine needle aspiration,pap smear, and cell culture. In preferred embodiments samples areobtained, e.g., in a biopsy, from cancerous tissue (e.g., a tumor) orfrom tissue that is suspected of being cancerous or of containing cancercells. In one particularly preferred embodiment samples are obtainedfrom lung tissue. In another preferred embodiment for testing epithelialcells for dysplasia, intra-epithelial pre-malignant changes, ormalignancy, the cells are obtained from the surface by gentle brushing,washing, or lavage to avoid obtaining p63-positive basal cells.

Non-human animals include, without limitation, laboratory animals suchas mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animalssuch as dogs and cats; and, farm animals such as sheep, goats, pigs,horses, and cows.

In preferred embodiments, the terms “about” and “approximately” shallgenerally mean an acceptable degree of error for the quantity measuredgiven the nature or precision of the measurements. Typical, exemplarydegrees of error are within 20 percent (%), preferably within 10%, andmore preferably within 5% of a given value or range of values.Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value, Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The invention provides for administration of a therapeutically effectivetreatment in conjunction with the diagnostic and prognostic methods. Theterm “therapeutically effective” refers to that amount of a treatmentregimen that is sufficient to result in a desired activity. Thus, asused to describe a cancer therapy, a therapeutically effective treatmentrefers to the amount of a chemotherapeutic compound or compositions,radiation, resection, or gene therapy, for example, that is sufficientto produce an effective outcome, such as tumor regression, increase timeof survival, increased time of remission, and the like.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction (for example, gastricupset, dizziness and the like) when administered to an individual.Preferably, and particularly where a vaccine is used in humans, the term“pharmaceutically acceptable” may mean approved by a regulatory agency(for example, the U.S. Food and Drug Agency) or listed in a generallyrecognized pharmacopeia for use in animals (for example, the U.S.Pharmacopeia).

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Sterile water or aqueous salinesolutions and aqueous dextrose and glycerol solutions are preferablyemployed as carriers, particularly for injectable solutions. Exemplarysuitable pharmaceutical carriers are described in “Reminington'sPharmaceutical Sciences” by E.W. Martin.

Detection of p63 Expression

A variety of methods can be employed for diagnostic and prognosticmethods using reagents. For example, using the methods described here itis possible to detect expression of a p63 nucleic acid or protein incells or tissues from an individual, such as in cells or tissues in asample (e.g., from a biopsy) obtained or derived from an individualsubject or patient. As explained above, p63 nucleic acids andpolypeptides are expressed at elevated levels in certain cancerous cellsand tissues including, for example, in the various types of cancer andtumor cells and tissues identified above, and not expressed in othertumor types.

Thus, using the methods described here (as well as other methods knownin the art) a skilled artisan may detect expression of p63 in a sampleof cells or tissue from an individual, and may thereby detect and/oridentify cells or tissue in that sample as being cancer cells or tissueof a particular pathological type. For example, in preferred embodimentsa skilled artisan may use such methods to identify cells or tissue in asample as being a particular type of cancer cell or tissue which isknown to express elevated levels of a p63 nucleic acid or polypeptide.Such cancer cells and tissues may be, for example, any of the particularcancer and tumor cell/tissue types described supra. In certain preferredembodiments the particular type of cancer cell or tissue identified insuch methods are lung cancer cells or tissue, such as cells or tissuesof bronchogenic carcinoma, lung carcinoma or small cell lung carcinoma.By using such methods to detect cancer cells or tissue in an individual,a skilled user may thereby diagnose the presence of the cancer in thatindividual. Similarly, the absence of p63 expression providesinformation about the nature of the cancer.

In preferred embodiments the methods described herein are performedusing pre-packaged diagnostic kits. Such kits may comprise at least onespecific p63 nucleic acid or a p63 specific antibody reagent. The kitand any reagent(s) contained therein may be used, for example, in aclinical setting, to diagnose patients exhibiting or suspected of havinga disorder such as a type of cancer.

A sample comprising a nucleated cell (of any cell type) from anindividual may be used in such diagnostic and prognostic methods as astarting source for genomic nucleic acid. and to detect mutations of ap63 gene. A sample comprising a cell of any cell type or tissue of anytissue type in which a p63 gene is expressed may also be used in suchdiagnostic methods, e.g., for detection of p63 gene expression or of p63gene products (such as p63 proteins), as well as for identifying cells,particularly cancer and tumor cells, that express or do not express p63.

Immunoassays

The diagnostic and prognostic methods of the invention include ones thatcomprise detecting levels of a p63 protein or other p63 polypeptides andincluding functionally conserved variants and fragments thereof. Forexample, antibodies directed against unimpaired, wild-type or mutant p63gene products or against functionally conserved variants or peptidefragments of a p63 gene product may be used as diagnostic and prognosticreagents, e.g., to detect various types of cancer cells and tumors. Suchreagents may be used, for example, to detect abnormalities in the levelof p63 gene product synthesis or expression. Antibodies and immunoassaymethods such as those described hereinbelow also have important in vitroapplications for assessing the efficacy of treatments, e.g., for cancer.Compounds that may have beneficial effects on a disorder associated withabnormal p63 expression (e.g., any of the types of cancer identifiedsupra) can be identified and a therapeutically effective dose for suchcompounds may be determined using such assays.

In vitro immunoassays can also be used to assess the efficacy ofcell-based gene therapy for a cancer or other disorder associated withabnormal p63 expression. For example, antibodies directed against p63polypeptides may be used in vitro to determine the level of p63 gene orpolypeptide expression achieved in tumor cells. Such methods may be usedto detect intracellular p63 gene products, preferably using whole cells,but also including cell lysates or extracts, to detect expression of p63gene products.

The isolated cells may be cells derived from cell culture or from anindividual (e.g., a biopsy sample from a patient suspected of having atype of cancer or other disorder associated with abnormal levels of p63expression, or suspected of having a propensity for such a cancer orother disorder).

As one example, antibodies or fragments of antibodies may be used todetect the presence of a p63 gene product, a variant of a p63 geneproduct or fragments thereof, for example, by immunofluorescencetechniques employing a fluorescently labeled antibody coupled with lightmicroscopic, flow cytometric or fluorimetric detection methods.

In particularly preferred embodiments, antibodies or fragments thereofmay also be employed histologically, for example in immunostaining,immunofluorescence or immunoelectron microscopy techniques, for in situdetection of a p63 gene product. In situ detection may be accomplishedby removing a histological specimen (e.g., a tissue sample) from apatient and applying thereto a labeled antibody of the present inventionor a fragment of such an antibody. The antibody or antibody fragment ispreferably applied by overlaying the labeled antibody or antibodyfragment onto a biological sample. Through the use of such a procedure,it is possible to detect, not only the presence of a p63 gene product,but also the gene product's distribution in the examined tissue. A widevariety of histological methods that are well known in the art (forexample, staining procedures) can be readily modified by those skilledin the art without undue experimentation to achieve such in situdetection.

Immunoassays for p63 gene products will typically comprise incubating abiological sample (for example, a biological fluid, a tissue extract,freshly harvested cells or cell lysates) in the presence of a detectablylabeled antibody that is capable of specifically binding a p63 geneproduct (including, for example, a functionally conserved variant or apeptide fragment thereof). The bound antibody may then be detected byany of a number of techniques well known in the art.

According to the invention, p63 polypeptide produced recombinantly or bychemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the p63 polypeptide. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library. The anti-p63antibodies of the invention may be cross reactive, e.g., they mayrecognize p63 from different species. Polyclonal antibodies have greaterlikelihood of cross reactivity. Alternatively, an antibody of theinvention may be specific for a single form of p63, such as murine p63.Preferably, such an antibody is specific for human p63. Antibodies foruse in the invention can also be specific for a specific isoform of p63,such as any of the three p63 splice forms, α, β, and γ. Since p63 formshave different cellular functions, it will be useful to develop a panelconsisting of an N-terminal-sequence specific antibody (that woulddetect only the full length p63 forms), and an antibody such as the oneused in this study, that recognizes both full length and truncated Δ-Nforms. Additional clinically useful differences in expression patternsare expected.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to p63 polypeptides or derivatives or analogsthereof. For the production of antibody, various host animals can beimmunized by injection with the p63 polypeptide, or a derivative (e.g.,fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the p63polypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, piuronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette—Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the p63polypeptide, or fragment, analog, or derivative thereof; any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(Nature 256:495-497, 1975), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983;Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96, 1985). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals(international Patent Publication No. WO 89/12690, published 28 Dec.1989). In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., J. Bacteriol.159:870, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda etal., Nature 314:452-454, 1985) by splicing the genes from a mouseantibody molecule specific for an p63 polypeptide together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in therapy ofhuman diseases or disorders (described infra), since the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, in particular an allergic response,themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778) can be adapted to produce p63polypeptide-specific single chain antibodies. Indeed, these genes can bedelivered for expression in vivo. An additional embodiment of theinvention utilizes the techniques described for the construction of Fabexpression libraries (Huse et al., Science 246:1275-1281, 1989) to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity for an p63 polypeptide, or its derivatives, oranalogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab fragmentswhich can be generated by reducing the disulfide bridges of the F(ab′)₂fragment, and the Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an p63 polypeptide, one may assay generatedhybridomas for a product which binds to an p63 polypeptide fragmentcontaining such epitope. For selection of an antibody specific to an p63polypeptide from a particular species of animal, one can select on thebasis of positive binding with p63 polypeptide expressed by or isolatedfrom cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the p63 polypeptide, e.g.,for Western blotting, imaging p63 polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc. using any of thedetection techniques mentioned above or known in the art.

In a specific embodiment, antibodies that agonize or antagonize theactivity of p63 polypeptide can be generated. Such antibodies can betested using the assays described infra for identifying ligands.

Molecular Biological Assays

The diagnostic and prognostic methods of the invention include methodsfor assaying the level of p63 gene expression. A variety of methodsknown in the art may be used to detect assay levels of p63 nucleic acidsequences in a sample. For example, RNA from a cell type or tissue, sucha tumor cell or tissue type, that is known or suspected to express thep63 gene may be isolated and tested utilizing hybridization or PCRtechniques known in the art. The isolated cells may be, for example,cells derived from a cell culture or from an individual. The analysis ofcells taken from a cell culture may be useful, e.g., to test the effectof compounds on the expression of a p63 gene, or alternatively, toverify that the cells are ones of a particular cell type that expressesa p63 gene.

As an example, and not by way of limitation, diagnostic methods for thedetection of p63 nucleic acids can involve contacting and incubatingnucleic acids (including recombinant DNA molecules, cloned genes ordegenerate variants thereof) obtained from a sample with one or morelabeled nucleic acid reagents, such as recombinant p63 DNA molecules,cloned genes or degenerate variants thereof, under conditions favorablefor specifically annealing or hybridizing these reagents to theircomplementary sequences in the sample nucleic acids. Preferably thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed or non-hybridizednucleic acids are removed. The presence of nucleic acids that havehybridized, if any such molecules exist, is then detected and the levelof p63 nucleic acid sequences to which the nucleic acid reagents haveannealed may be compared to the annealing pattern or level expected froma control sample (e.g., from a sample of normal, non-cancerous cells ortissues) to determine whether p63 nucleic acid is expressed at anelevated level.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.509.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook at al., supra, 11.7-11.8). A minimumlength for a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides; and more preferably the lengthis at least about 20 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) 65° C. In a specific embodiment, “high stringency”refers to hybridization and/or washing conditions at 68° C. in 0.2×SSC,at 42° C. in 50% formamide, 4×SSC, or under conditions that affordlevels of hybridization equivalent to those observed under either ofthese two conditions.

In a preferred embodiment of such a detection scheme, the nucleic acidfrom the cell type or tissue of interest may be immobilized, forexample, to a solid support such as a membrane or a plastic surface (forexample, on a nylon membrane, a microtiter plate or on polystyrenebeads). After incubation, non-annealed, labeled p63 nucleic acidreagents may be easily removed and detection of the remaining, annealed,labeled p63 nucleic acid reagents may be accomplished using standardtechniques that are well-known in the art.

Alternative diagnostic methods for the detection of p63 nucleic acids inpatient samples or in other cell or tissue sources may involve theiramplification, e.g., by PCR (see, for example, the experimentalembodiment taught in U.S. Pat. No. 4,683,202) followed by detection ofthe amplified molecules using techniques that are well known to those ofskilled in the art. The resulting level of amplified p63 nucleic acidmay be compared to those levels that would be expected if the samplebeing amplified contained only normal levels of p63 nucleic acid, asnormal cells or tissues, to determine whether elevated levels of a p63nucleic acid are expressed.

In one preferred embodiment of such a detection scheme, a cDNA moleculeis synthesized from an RNA molecule of interest (e.g., by reversetranscription). A sequence within the cDNA may then be used as atemplate for a nucleic acid amplification reaction such as PCR. Nucleicacid reagents used as synthesis imitation reagents (e.g., primers) inthe reverse transcription and amplification steps of such an assay arepreferably chosen from the p63 nucleic acid sequences described hereinor are fragments thereof. Preferably, the nucleic acid reagents are atleast about 9 to 30 nucleotides in length. The amplification may beperformed using, e.g., radioactively labeled or fluorescently labelednucleotides, for detection. Alternatively, enough amplified product maybe made such that the product can be visualized by standard ethidiumbromide or other staining methods.

p63 gene expression assays of the invention may also be performed insitu (i.e., directly upon tissue sections of patient tissue, which maybe fixed and/or frozen), thereby eliminating the need of nucleic acidpurification. p63 nucleic acid reagents may be used as probes or asprimers for such in situ procedures (see, for example, Nuovo, PCR InSitu Hybridization: Protocols And Application, 1992, Raven Press, NewYork). Alternatively, if a sufficient quantity of the appropriate cellscan be obtained, standard Northern analysis can be performed todetermine the level of p63 gene express by detecting levels of p63 mRNA.

EXAMPLES

The present invention may be better understood by reference to thefollowing examples, which are provided by way of illustration and arenot limiting.

Example 1 Immunohistochemical Study of Expression of p53-homolog p63 inPulmonary Neoplasms

p63 is a nuclear protein recently discovered in a screen for genes withhomology to the tumor suppressor p53 gene. This Example describes p63expression in benign lung tissue and neoplasms of pulmonary origin.

Materials and Methods

Tissues. Archival, routinely processed, formalin-fixed,paraffin-embedded surgical pathology specimens from bronchoscopic biopsyand pneumonectomy and/or lobectomy specimens were examined.

Antibody. The antibody used in this study was anti-p63 monoclonalantibody 4A4 (Santa Cruz Biotechnology, Inc., Santa Cruz Calif.). Thisantibody is reactive against a region common to both the full-length andtruncated forms of p63.

Immunohistochemical staining. Five-micron-thick sections were heated anddeparaffinized and were treated with 0.3% hydrogen peroxide to blockendogenous peroxidase activity and pretreated with citric acid (pH 6.0)for antigen retrieval. Slides were then incubated overnight at roomtemperature with an anti-p63 antibody and stained using a streptavidinbiotin-based immunoperoxidase staining kit (BioGenex, San Ramon, CA),according to the manufacturer's instructions. The kit employsdiaminobenzidine as chromagen, and then counterstain with hematoxylinand/or eosin,

p63 expression was considered positive only if distinct nuclear stainingwas present. Intense positive staining of bronchial reserve cell nucleiwas noted as an internal positive control for p63 staining of histologicsections.

Results

Normal epithelium. In normal lung, p63 intensely stained nuclei ofbronchial reserve cells but did not stain ciliated cells, alveolarepithelial cells or non-epithelial cells. The lower strata of squamousmetaplastic bronchial epithelium stained positively for p63.

Squamous cell carcinoma. Twenty-six out of twenty-seven cases (96%) ofsquamous cell carcinoma stained positively for p63. Inwell-differentiated carcinomas staining was generally inverselyproportional to the degree of differentiation or keratinization asassessed by counterstaining slides with eosin. Staining, noted inbasilar peristromal areas, diminished as cells became keratinized. Inmany keratinizing carcinomas, p63 expression was lost with onset ofkeratinization. However, in some cases, we noted keratinization withpersisting p63 positivity, suggesting relative loss of cell regulatorysuppression of proliferation concommitant with differentiation. Poorlydifferentiated carcinomas showed very high proportions (80-100%) ofp63-positive nuclei.

Bronchioloalveolar carcinoma. Eight out of eight cases (100%) showed nodetectable staining of tumor cell nuclei.

Adenocarcinoma. Staining of adenocarcinomas was variable: 9 out of 19tumors showed no detectable staining. One out of 4 adenocarcinomas withalveolar spread showed positive staining.

Adenosquanous carcinomas. Two out of 3 adenosquanios carcinomas revealeda unique basalar staining pattern that approximated the pattern innormal ciliated bronchial epithelium.

Small cell undifferentiated carcinoma. Ten out of ten cases (100%)showed no detectable staining of tumor cell nuclei.

Carctinoid. Six out of six cases were almost entirely negative. Rarepositive cells were of indeterminate, probably non-neoplastic origin andappeared niorphologically distinct from the tumor cells. Large cellcarcinoma. Eight out of 12 tumors stained positively for p63.

Discussion

p63 is a p53 homolog postulated to play a role in stem cell commitmentin squamous epithelia. The activation of cell pathways that suppresscell division is essential for the commitment of undifferentiateddividing basalar cells to undergo maturation, Consistent with the knownrole of p53 as an activator of such suppressive pathways in cellsresponding to DNA damage, p63 might serve in a homologous physiologicrole in normal stem cell commitment.

Accumulated data from several laboratories demonstrates expression ofp63 in normal human cervical epidermis, hair follicles, and instratified epidermal cultures, p63 protein is primarily restricted tocells with high proliferative potential and is absent from the cellsthat are undergoing terminal differentiation. In squamous cellcarcinomas of the epidermis, the number of cells containing p63 andtheir distribution was found to depend on the degree of anaplasia.(Parsa et al., J. Invest. Dermatol. 1999, 113:1099-1105). In highlydifferentiated tumors, p63 was confined to a ring of basal-like cellssurrounding, but at a distance from, centers of terminaldifferentiation. In less differentiated tumors, most cells contained p63and their distribution was chaotic with respect to centers of terminaldifferentiation (Yamaguchi et al, Int. J. Cancer 2000, 86:684-9). FISHanalysis shows amplification of the locus containing the p63 gene inprimary head and neck carcinoma and in cervical carcinoma.

The results of the current study indicate that p53 homolog p63 stainspoorly differentiated squamous cell carcinomas in a consistent anduniform manner with very high percentages (approaching 100%) of tumorcells showing nuclear staining. Staining in well differentiated squamouscell carcinomas is also observed to be strong but is apparent inbasalar, para-stromal areas lacking keratinization. Staining of otherlung tumor types revealed no staining in undifferentiated small-cellcarcinomas and bronchioloalveolar carcinomas, and essentially negligiblestaining in carcinoids.

Adenocarcinomas showed varied staining patterns. When positive, thepatterns differed from the staining pattern seen in squamous carcinomas.

One highly unusual pattern was that seen in two cases of adenosquamouscarcinoma. The similarity of the unique basalar staining pattern to thestaining of reserve cells in normal bronchial epithelium raises thepossibility that this might be a form of adenocarcinoma in situ.

p63 expression appears to differ between bronchioloalveolar carcinomaand adenocarcinoma with alveolar spread: no cases of the former stainedpositively, whereas 1 out of 4 cases of the latter were p63-positive.

We have noted in other studies p63 staining in myoepithelial cells ofbreast; expression in thyroid follicular epithelial cells in Hashimoto'sthyroiditis, and expression in a spectrum of other neoplasms. Theseinclude papillary thyroid carcinoma, urothelial carcinoma, endometrialadenocarcinoma and endocervical adenocarcinoma (see Examples 2 and 3,infra). Of potential significance, all of these tumors arising fromnon-squamous epithelia have the capacity to undergo squamousdifferentiation, and all are capable of expressing p63. In contrast,renal cell carcinomas, which never undergo squamous differentiation, donot express p63.

Ascertaining the specific tumor type in primary lung cancer hasimportant implications, both for treatment and prognosis. Small cellundifferentiated carcinoma differs from the other histologic types inthat it is a more aggressive tumor with a greater capacity fordissemination, therefore having a worse prognosis. For this reason, andbecause of greater chemosensitivity, the treatment of patients withsmall cell undifferentiated carcinoma is primarily chemotherapy.Surgery, on the other hand, is the therapy of choice for the remainingnon-small cell lung carcinomas.

Distinction of poorly differentiated squamous carcinoma from small cellundifferentiated carcinoma can be difficult, particularly in small tumorsamples found in cytopathologic specimens and in bronchoscopic biopsies.Previous studies have reported that the accuracy of diagnosis onbronchial brush specimens was 62 to 97.5% and specifically 53.8% forsmall cell undifferentiated carcinoma. Hence, using immunohistochemistryto determine the presence of p63 in cytologic specimens, which willdistinguish a poorly-differentiated squamous cell carcinoma from a smallcell undifferentiated carcinoma has evident clinical utility.

Example 2 Expression of p63 Protein in Subtypes of Transitional Cell andRenal Cell Carcinomas

The p53 family of tumor suppressor genes includes p63, which is highlyexpressed in the basal layers of epithelial tissues. P63 may eitherpromote apoptosis or antagonize p53, depending upon the expressedprotein isoform. Expression of p63 has been found in transitionalepithelium. Expression of p63 in transitional cell carcinomas wascompared with expression in renal cell carcinomas. Non-cancerous tissuesassociated with the urinary tract were also examined.

Methods

Formalin-fixed, paraffin-embedded archival tissue from 81 patients wasimmunostained with p63 monoclonal antibody 4A4 (Santa Cruz) reactiveagainst all subtypes of p63 protein as described above for Example 1.Immunostaining patterns and routine H&E staining patterns wereinterpreted by four observers. Nuclear staining was considered positive.

Results

Normal tissue. Benign adrenal cortex (1), prostate (2), and other benignkidney specimens were negative for p63 staining.

Transitional cell carcinomas. 96% of transitional cell carcinomasstained diffusely for p63, (low grade bladder 9/9, high grade bladder10/22, ureter 2/2, renal pelvis 8/8). Bladder carcinoma-in-situ casesall stained (n=18) positively to varying degrees, including basal (about½ of cases), diffuse, and rare. Cystitis cystica with focal dysplasiashowed strong basal staining (1/1) for p63.

Renal cell carcinomas, In contrast all renal cell carcinoma specimens(21/21), regardless of type and grade, (including clear cell, papillary,chromophobe, and collecting duct) were negative as werexanthogranulomatous pyelonephritis (1).

Conclusion

Staining of p63 is strongly positive in transitional cell carcinomas anduniformly negative in renal cell carcinomas. This sharp distinction maybe a useful diagnostic tool in preoperative evaluation and therapeuticplanning in patients with upper urinary tract lesions.

Example 3 Expression of p63 in Papillary Thyroid Carcinoma and inHashimoto's Thyroiditis

Nuclear proteins were discovered as a consequence of a screen forp53-homologous genes. Thus far, p63 expression has been demonstrated inbasal cells of squamous epithelium and in urothelium.Immunochistochemical detection of p63 expression was evaluated in normaland abnormal thyroid tissue,

Materials and Methods

Five micron sections from routinely fixed and processed archival thyroidresection specimens were pretreated with citric acid pH 6.0 for antigenretrieval at 100° C. for 5 minutes, then incubated overnight withanti-p63 monoclonal antibody 4A4 (Santa Cruz). Slides were stained usinga streptavidinbiotin kit (BioGenex), followed by reaction withdiaminobenzidine, and then counterstaining with hematoxylin.

Results

Conditions negative for p63. No p63 expression was seen in normalthyroid tissue, twelve follicular adenomata, eleven Hurthie celladenomata, six medullary carcinomas, two anaplastic carcinomas, and sixHurthle cell carcinomas. There was rare staining in one of sevenfollicular carcinomas. In addition, there was no p63 staining in tennodular goiter samples, and one of 8 Grave's disease samples stained.

Hashimoto's thyroiditis. In contrast, seventy five percent (12/16) ofHashimoto's thyroiditis cases and seventy six percent (22/29) ofpapillary thyroid carcinomas showed epithelial cells with nuclearpositivity for p63. Staining of tumor cells ranged from strong and/orfrequent to weak and/or uncommon. In Hashimoto's cases, staining wascharacteristically intense but limited to several follicles. Of note, onparallel sections, CK19 positivity was found in p63-positive carcinomasbut also in numerous benign follicles within areas of p63-positivethyroiditis.

Conclusions

p63 is expressed in papillary thyroid carcinoma and in Hashimoto'sthyroiditis. Considering the debated association of papillary thyroidcarcinoma with Hashimoto's thyroiditis, it is possible that p63 may be apotential pathobiologic link between the two disorders.

p63 may also be of use in distinguishing papillary carcinoma from otherthyroid neoplasms, particularly on fine needle aspirates. Thisobservation has particular relevance for avoiding unnecessary surgerybecause of a misdiagnosis or the inability to diagnose nodular goiterinstead of papillary carcinoma.

Example 4 Detecting p63 in Non-Traumatically Obtained Samples

In this example, we describe results of examination of pre-canceroussquamous intra-epithelial lesions of the uterine cervix compared withnormal cervical epithelium.

Methods

Cervical and vaginal epithelial cells were obtained from gentle swabbingof the uppermost layers and subjected to immunostaining for p63 asdescribed infra.

Results

In contrast to normal epithelium, in which p63 is expressed in the lowercell layers of the epithelium, in accordance with findings by others,p63 expression is found in the uppermost layers of high grade squamousintraepithelial lesions.

Discussion

Since the papanicolaou “pap” smear of the cervical and vaginalepithelium involves sampling of the uppermost cell layers of theseepithelia via gentle, non-traumatic swabbing, brushing or othernon-traumatic sampling means, detection of p63 alone, or in a multiplexpanel with other markers, in cells from cervix or vaginal squamousepithelium can be used to signify the presence of a clinically advancedsquamous intraepithelial lesion. This can be adapted for automatedscreening for aberrant expression of p63 in cells from the uppermostlayers of cervical-vaginal squamous epithelium in cervical-vaginalsmears, as a substitute for manual screening presently performed onmillions of pap smears to detect such lesions,

Similarly, detection of cancer or pre-cancerous changes by detecting p63(at the gene, mRNA, or protein level) in a cell or fluid samplefollowing non-traumatic sampling (such as lavage, gentle brush, wash) ofother epithelia in which abnormal p63-positive epithelia replace normalepithelia (the uppermost layer of cells of which are normallyp63-negative) constitutes another claim of this patent. This applies notonly to cervical vaginal sampling, but to sampling of oral, upperrespiratory tract, lower respiratory tract, esophageal, cyst contentsamples or cyst lining, urinary tract, endocervical, endometrial, andother surfaces or mucosae.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1-24. (canceled)
 25. A method for distinguishing a squamous cell tumoror a tumor of epithelial cells with squamous cell potential from anon-epithelial cell tumor or a glandular carcinoma without squamousdifferentiation or potential, which method comprises: testing a samplesuspected of containing a tumor for p63 expression; and classifying thesample as positive or negative for p63 expression, wherein a sample isconsidered positive only if the sample exhibits nuclear concentrationsof p63, and wherein positive p63 expression indicates that the samplecontains a squamous cell tumor or a tumor of epithelial cells withsquamous cell potential and the absence of p63 expression indicates thatthe tumor is a non-epithelial tumor or a glandular tumor withoutsquamous differentiation or potential.
 26. The method according to claim25, wherein the glandular carcinoma is a renal carcinoma.
 27. A methodfor distinguishing a squamous cell tumor or a tumor of squamousepithelia, transitional cells, or glandular epithelia with squamous cellpotential from a non-epithelial cell tumor or a tumor without squamousdifferentiation or potential, which method comprises: testing a samplesuspected of containing a tumor for p63 expression; and classifying thesample as positive or negative for p63 expression, wherein a sample isconsidered positive only if the sample exhibits nuclear concentrationsof p63, and wherein positive p63 expression indicates that the samplecontains a squamous cell tumor or a tumor of squamous epithelia,transitional cells or glandular cells with squamous cell potential andthe absence of p63 expression indicates that the tumor is anon-epithelial tumor or a tumor without squamous differentiation orpotential.
 28. The method according to claim 25 wherein testing for p63expression comprises detecting p63 protein.
 29. The method according toclaim 28, wherein testing for p63 protein expression comprises detectingthe p63 protein with an immunoassay.
 30. The method of claim 29, whereinthe immunoassay is an immunohistochemical assay.
 31. A method ofdistinguishing a urothelial carcinoma from a non-epithelial cell tumoror a tumor without squamous differentiation or potential, which methodcomprises: testing a sample suspected of containing a tumor for p63expression; and classifying the sample as positive or negative for p63expression, wherein a sample is considered positive only if the sampleexhibits nuclear concentrations of p63, and wherein positive p63expression indicates that the sample contains a urothelial carcinoma andthe absence of p63 expression indicates that the tumor is anon-epithelial tumor or a tumor without squamous differentiation orpotential.
 32. A method of distinguishing a lung adenocarcinoma from alung squamous carcinoma, comprising identifying a lung sample suspectedof containing said lung adenocarcinoma or said lung squamous carcinomaas comprising tumor cells that are positive for p63 expression; anddetermining whether said tumor cells exhibit a squamous carcinoma p63staining pattern, wherein p63 expression exhibiting said squamouscarcinoma p63 staining pattern in said tumor cells indicates that saidlung sample contains said lung squamous carcinoma and p63 expression insaid tumor cells that does not exhibit said squamous carcinoma p63staining pattern indicates that said lung sample contains said lungadenocarcinoma.
 33. The method of claim 32 wherein said squamouscarcinoma p63 staining pattern is a uniform nuclear staining ofsubstantially all of said tumor cells.
 34. The method of claim 32wherein said squamous carcinoma p63 staining pattern is strong stainingapparent in basalar, para-stromal areas lacking keritinization.
 35. Themethod according to claim 27 wherein testing for p63 expressioncomprises detecting p63 protein.
 36. The method according to claim 35,wherein testing for p63 protein expression comprises detecting the p63protein with an immunoassay.